A large segment of the scientific community is involved in discovering more about the structure and function of the human immunological response and pathogen defense systems including those conditions such as, for example, acquired immune deficiency syndrome and associated diseases brought about by infections with opportunistic pathogens which may effect or compromise those systems. As part of this investigation, a number of inquiries have been made into the biochemistry of the immune or defense systems of other creatures, both as models upon which to base theory, and as a source of potentially transplantable factors.
One such group of immune or defense factors are the antimicrobial peptides first reported in 1987 by two groups of researchers, one headed by Dudley Williams and one headed by Michael Zasloff. These groups successfully characterized and reported a number of peptides which are secreted by the glands contained within the skin of the African Clawed Frog, Xenopus laevis which appear to have antimicrobial activity. See, Giovannini, et al., "Biosynthesis and Degradation of Peptides Derived from Xenopus laevis Prohormones" Biochem. J. 243, (1987), 113-120; and Zasloff, "Magainins, a Class of Anti-Microbial Peptides from Xenopus Skin: Isolation, characterization of two active forms and partial cDNA sequence of a precursor", Proc. Natl. Acad. Sci. (USA) 84, (1987), 5449-5453, and Terry et al., "The cDNA Sequence Coding for Prepro-PGS (Prepro-Magainins) and Aspects of the Processing of This Prepro-Polypeptide," J. Biol. Chemistry, 263, (1988), 5745-5751. Their research was prompted, at least in part, by the observation that this species of frog has remarkable recuperative power and the ability to remain free from infection during wound-healing with little or no post operative care. These peptides are collectively referred to as magainins.
The published works regarding magainins and other classes of antibiotic or antimicrobial peptides (for example, cecropins, defensins, sarcotoxins, melittins, and the like) of which the inventors are aware have generally centered on human pharmaceutical-related health technologies. Exceptions, however, include two patent applications filed by Jaynes et al., (WO 89/04371 and WO 88/00976) which generally relate to plants which have been genetically enhanced for disease resistance. Jaynes et al. have speculated without supporting data that genetically transformed plants may be produced which contain an expressible heterologeous gene for an antimicrobial peptide. In this way, it is hoped that the plant has enhanced resistance to disease. According to Jaynes et al., however, peptides such as melittins, bombinins, and magainins having less than about 30 residues are not preferred for use in crop protection applications.
Others have published information relating to the existence of antimicrobial peptides in plants or, in fact, the use of antimicrobial peptides to protect plants from plant pathogens. See, EPO 0,299,828; P. Casteels et al., "Apidaecins: Antibacterial Peptides From Honeybees," The EMBO J. 8, (1989), 2387-2391; F. Ebrahim-Nesbat et al., "Cultivar-Related Differences in the Distribution of Cell-Wall-Bound Thionins in Compatible and Incompatible Interactions Between Barley and Powdery Mildew," Planta 179, (1989), 203-210. Most of this work centered upon the identification and use, either in plants or animals, of basically full-length natural antimicrobial peptides.
In addition to this research, a number of variations on naturally occurring peptides have been investigated. Specifically, a number of magainin based derivatives having varying degrees of activity have been produced and investigated. See Juretic, et al., "Magainin 2 Amide and Analogues, Antimicrobial Activity, Membrane Depolarization and Susceptibility of Proteolysis," Febs Lett. 249, (1989), 219-223; Chen, et al, "Synthetic Magainin Analogues With Improved Antimicrobial Activity," Febs Lett. 236, (1988), 462-466; Chen et al, U.S. patent application Ser. No. 280,363, filed Dec. 6, 1988; Cuervo, et al., "Synthesis and Antimicrobial Activity of Magainin Alanine Substitution Analogs," Proceedings of the Eleventh American Peptide Symposium; Peptides: Chemistry, Structure and Biology (J. E. Rivier, et al.), (1990), pp. 124-126, published by ESCOM-Leiden, Neth.; Cuervo, et al., "The Magainins: Sequence Factors Relevant to Increased Antimicrobial Activity and Decreased Hemolytic Activity," Peptide Research 1, (1988), 81-86; World Patent Application No. WO 88/06597; and Japanese Patent Application No. JP-1/299,299. These include the complete single residue omission analogue series of Magainin 1 and 2, select N-terminal omissions of Magainin 2, as well as the complete alanine (Ala) replacement analog series of Magainin 2, and Magainin 2 derivatives which may be useful as an antibiotic and/or an anti-cancer drug and which are substituted at the 5th and 12th positions.
Another such investigation was conducted by Bascomb et al. which is reported in U.S. patent application Ser. No. 566,152 filed on Aug. 10, 1990, the complete text of which is hereby incorporated by reference. Bascomb et al. identified specific positions on natural magainins that are important for activity, proteolytic sensitivity to at least one plant protease, and/or phytotoxicity. Bascomb et al. also developed strategic amino acid substitutions and/or deletions relating thereto.
The aforementioned efforts are far from complete. They have not, as yet, provided a complete picture of even the simplest model for relating antibiotic peptide structure to function. Therefore, there still remains a need for further investigation into antimicrobial peptides and their uses. Furthermore, in light of the promising information provided by those including Bascomb et al. who have modified natural peptide structures, further investigation into the effects of various modified forms of naturally occurring peptides may provide both useful models for further investigation and, indeed, useful peptides for combatting plant and animal microbial pathogens.
The present invention is directed to several discoveries. First, the present inventors have identified certain new peptides and their functional derivatives including reverse antimicrobial peptides which are active against at least one microbial pathogen and, preferably, at least one plant pathogen. They have also discovered the usefulness of the non-amide form of a previously identified compound (PGL.sup.a), known herein as PGL.sup.c. The inventors have also discovered oligopeptides having antimicrobial activity, and that these peptides and oligopeptides, as well as peptides such as, for example, cecropin P1 (herein termed P1 since it is of porcine origin, not insect origin), are useful in providing protection against plant pathogens, either alone or when included within mixtures. These peptides or their functional derivative peptides may additionally possess decreased phytotoxicity and/or decreased sensitivity to proteolytic degradation. Thus, these compounds and certain mixtures thereof are useful for providing protection against at least one plant pathogen and, may indeed have broader application into animals and foodstuffs.